Method for determining the nucleotide base sequence of a DNA molecule

Abstract

This invention relates to T7-type DNA polymerases and methods for using them.

We claim: 
     
       1. A method for determining the nucleotide base sequence of a DNA molecule, comprising the steps of: annealing said DNA molecule with a labelled primer molecule able to hybridize to said DNA molecule,   incubating separate portions of the annealed mixture in at least four vessels, each vessel containing four different deoxynucleoside triphosphates, a DNA polymerase except reverse transcriptase, and a chain terminating agent which terminates DNA synthesis at a specific nucleotide base, wherein each agent terminates synthesis at a different nucleotide base, and wherein the concentration of all four deoxynucleoside triphosphates at the start of said incubating is sufficient to allow DNA synthesis to continue until terminated by said agent, and   separating the DNA products of each incubating reaction according to their size, whereby at least a part of the nucleotide base sequence of said DNA molecule can be determined.   
     
     
       2. A method for determining the nucleotide base sequence of a DNA molecule, comprising the steps of: annealing said DNA molecule with a primer molecule able to hybridize to said DNA molecule in a single vessel;   incubating the annealed mixture with a DNA polymerase except reverse transcriptase in a single vessel containing four different deoxynucleoside triphosphates, at least one said deoxynucleoside triphosphate being labelled, under conditions in which said primer can be extended,   incubating separate portions of the extended primer mixture in at least four vessels, each vessel containing four different deoxynucleoside triphosphates, and a chain terminating agent which terminates DNA synthesis at a specific nucleotide base, wherein each agent terminates synthesis at a different nucleotide base, and wherein said deoxynucleoside triphosphates are at a concentration sufficient to allow DNA synthesis to continue until terminated by said agent, and   separating the DNA products of each incubating reaction according to their size, whereby at least a part of the nucleotide base sequence of said DNA molecule can be determined.   
     
     
       3. A method for determining the nucleotide base sequence of a DNA molecule, comprising the steps of: annealing said DNA molecule with a labelled primer molecule able to hybridize to said DNA molecule.   incubating the annealed mixture in the presence of four different deoxynucleoside triphosphates, a DNA polymerase except reverse tanscriptase, and a chain terminating agent which terminates DNA synthesis at a specific nucleotide base, wherein the concentration of all four deoxynucleoside triphosphates is sufficient to allow DNA synthesis to continue until terminated by said agent, and   separating the DNA products of said incubating reaction according to their size, whereby at least a part of the nucleotide base sequence of said DNA molecule can be determined.   
     
     
       4. A method for determining the nucleotide base sequence of a DNA molecule, comprising the steps of: annealing said DNA molecule with a primer molecule able to hybridize to said DNA molecule;   incubating the annealed mixture with a DNA polymerase except reverse transcriptase in the presence of four different deoxynucleoside triphosphates, at least one said deoxynucleoside triphosphate being labelled, under conditions in which said primer can be extended,   incubating the extended primer mixture in the presence of four different deoxynucleoside triphosphates, and a chain terminating agent which terminates DNA synthesis at a specific nucleotide base, wherein said deoxynucleoside triphosphates are at a concentration sufficient to allow DNA synthesis to continue until terminated by said agent, and   separating the DNA products of each incubating reaction according to their size, whereby at least a part of the nucleotide base sequence of said DNA molecule can be determined.   
     
     
       5. The method of claim 1 or 2 wherein said DNA polymerase is a processive DNA polymerase. 
     
     
       6. The method of claim 5 wherein said DNA polymerase is a T7-type DNA polymerase. 
     
     
       7. The method of claim 6 wherein said T7-type DNA polymerase is T7 DNA polymerase. 
     
     
       8. The method of claim 1 or 2 wherein said polymerase has a level of exonuclease activity which is sufficiently low to permit the nucleotide base sequence of the DNA molecule to be determined. 
     
     
       9. The method of claim 8 wherein said DNA polymerase has less than 10% of the exonuclease activity of the naturally associated level of exonuclease activity of said polymerase. 
     
     
       10. The method of claim 9 wherein said DNA polymerase has less than 1% of the exonuclease activity of the naturally associated level of exonuclease activity of said polymerase. 
     
     
       11. The method of claim 5 wherein said DNA polymerase remains bound to said DNA molecule for at least 500 bases before dissociating. 
     
     
       12. The method of claim 11 wherein said polymerase remains bound to said DNA molecule for at least 1,000 bases before dissociating. 
     
     
       13. The method of claim 6 wherein said polymerase is substantially the same as that in cells infected with a T7-type phage. 
     
     
       14. The method of claim 6 wherein said T7-type phage is T7, T3, DI, DIII, H, W31, gh-1, Y, A1122 or Sp6. 
     
     
       15. The method of claim 1, or 2 wherein said polymerase is non-discriminating for dideoxy nucleotide analogs. 
     
     
       16. The method of claim 1 or 2 wherein said DNA polymerase has less than 500 units of exonuclease activity per mg of DNA polymerase. 
     
     
       17. The method of claim 16 wherein said DNA polymerase has less than 50 units of exonuclease activity per mg of DNA polymerase. 
     
     
       18. The method of claim 17 wherein said DNA polymerase has less than 1 unit of exonuclease activity per mg of DNA polymerase. 
     
     
       19. The method of claim 18 wherein said DNA polymerase has no detectable exonuclease activity. 
     
     
       20. The method of claim 1 or 2 wherein said DNA polymerase is able to utilize primers of 10 base pairs or more. 
     
     
       21. The method of claim 20 wherein said polymerase is able to utilize primers of 4 base pairs or more. 
     
     
       22. The method of claim 1 or 2 wherein said terminating agent is a dideoxynucleotide. 
     
     
       23. The method of claim 3 wherein said primer is labelled.